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- EMDB-29977: AP2 bound to an MSP1 nanodisc -

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Basic information

Entry
Database: EMDB / ID: EMD-29977
TitleAP2 bound to an MSP1 nanodisc
Map dataFull map
Sample
  • Complex: AP2 bound to MSP2N2 nanodisc
    • Protein or peptide: AP-2 complex subunit alpha-2
    • Protein or peptide: AP-2 complex subunit beta
    • Protein or peptide: AP-2 complex subunit mu
    • Protein or peptide: AP-2 complex subunit sigma
    • Protein or peptide: MSP1
Keywordsclathrin-dependent endocytosis / peripheral membrane protein / ENDOCYTOSIS
Function / homology
Function and homology information


Formation of annular gap junctions / Gap junction degradation / LDL clearance / WNT5A-dependent internalization of FZD2, FZD5 and ROR2 / WNT5A-dependent internalization of FZD2, FZD5 and ROR2 / LDL clearance / WNT5A-dependent internalization of FZD4 / VLDLR internalisation and degradation / Retrograde neurotrophin signalling / Trafficking of GluR2-containing AMPA receptors ...Formation of annular gap junctions / Gap junction degradation / LDL clearance / WNT5A-dependent internalization of FZD2, FZD5 and ROR2 / WNT5A-dependent internalization of FZD2, FZD5 and ROR2 / LDL clearance / WNT5A-dependent internalization of FZD4 / VLDLR internalisation and degradation / Retrograde neurotrophin signalling / Trafficking of GluR2-containing AMPA receptors / Retrograde neurotrophin signalling / clathrin coat / clathrin adaptor complex / WNT5A-dependent internalization of FZD4 / extrinsic component of presynaptic endocytic zone membrane / VLDLR internalisation and degradation / cardiac septum development / MHC class II antigen presentation / Recycling pathway of L1 / AP-2 adaptor complex / regulation of vesicle size / postsynaptic neurotransmitter receptor internalization / Recycling pathway of L1 / clathrin coat assembly / Cargo recognition for clathrin-mediated endocytosis / Cargo recognition for clathrin-mediated endocytosis / positive regulation of synaptic vesicle endocytosis / Clathrin-mediated endocytosis / clathrin adaptor activity / Clathrin-mediated endocytosis / vesicle budding from membrane / membrane coat / clathrin-dependent endocytosis / MHC class II antigen presentation / neurotransmitter receptor internalization / positive regulation of protein localization to membrane / coronary vasculature development / signal sequence binding / negative regulation of protein localization to plasma membrane / aorta development / regulation of hematopoietic stem cell differentiation / ventricular septum development / low-density lipoprotein particle receptor binding / clathrin binding / protein serine/threonine kinase binding / positive regulation of endocytosis / Trafficking of GluR2-containing AMPA receptors / positive regulation of receptor internalization / synaptic vesicle endocytosis / vesicle-mediated transport / Neutrophil degranulation / phosphatidylinositol binding / secretory granule / kidney development / intracellular protein transport / cytoplasmic side of plasma membrane / kinase binding / disordered domain specific binding / synaptic vesicle / heart development / cytoplasmic vesicle / postsynapse / protein-containing complex assembly / transmembrane transporter binding / protein domain specific binding / intracellular membrane-bounded organelle / glutamatergic synapse / lipid binding / synapse / protein-containing complex binding / protein kinase binding / mitochondrion / plasma membrane
Similarity search - Function
AP-2 complex subunit sigma / Clathrin adaptor, alpha-adaptin, appendage, C-terminal subdomain / Adaptor protein complex AP-2, alpha subunit / Alpha adaptin AP2, C-terminal domain / Mu2, C-terminal domain / AP-2 complex subunit mu, N-terminal / Adaptor protein complex, sigma subunit / Clathrin adaptor, beta-adaptin, appendage, Ig-like subdomain / Beta-adaptin appendage, C-terminal subdomain / AP-1/2/4 complex subunit beta ...AP-2 complex subunit sigma / Clathrin adaptor, alpha-adaptin, appendage, C-terminal subdomain / Adaptor protein complex AP-2, alpha subunit / Alpha adaptin AP2, C-terminal domain / Mu2, C-terminal domain / AP-2 complex subunit mu, N-terminal / Adaptor protein complex, sigma subunit / Clathrin adaptor, beta-adaptin, appendage, Ig-like subdomain / Beta-adaptin appendage, C-terminal subdomain / AP-1/2/4 complex subunit beta / Beta2-adaptin appendage, C-terminal sub-domain / Beta2-adaptin appendage, C-terminal sub-domain / AP complex subunit beta / Clathrin adaptor complex, small chain / Clathrin adaptor complexes small chain signature. / Clathrin adaptor complexes medium chain signature 1. / Clathrin adaptor, mu subunit / Clathrin adaptor, mu subunit, conserved site / Clathrin adaptor complexes medium chain signature 2. / Coatomer/calthrin adaptor appendage, C-terminal subdomain / Adaptor complexes medium subunit family / AP complex, mu/sigma subunit / Clathrin adaptor complex small chain / AP-2 complex subunit mu, C-terminal superfamily / Mu homology domain / Mu homology domain (MHD) profile. / Clathrin adaptor, alpha/beta/gamma-adaptin, appendage, Ig-like subdomain / Adaptin C-terminal domain / Adaptin C-terminal domain / Clathrin/coatomer adaptor, adaptin-like, N-terminal / Adaptin N terminal region / Clathrin adaptor, appendage, Ig-like subdomain superfamily / Longin-like domain superfamily / TBP domain superfamily / Armadillo/beta-catenin-like repeats / Armadillo / Armadillo-like helical / Armadillo-type fold
Similarity search - Domain/homology
AP-2 complex subunit alpha-2 / AP-2 complex subunit sigma / AP-2 complex subunit mu / AP-2 complex subunit beta
Similarity search - Component
Biological speciesMus musculus (house mouse) / Homo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 4.2 Å
AuthorsSarsam RD / Cannon KS / Baker RW
Funding support1 items
OrganizationGrant numberCountry
Not funded
CitationJournal: J Struct Biol / Year: 2023
Title: Lipid nanodiscs as a template for high-resolution cryo-EM structures of peripheral membrane proteins.
Authors: Kevin S Cannon / Reta D Sarsam / Tanita Tedamrongwanish / Kevin Zhang / Richard W Baker /
Abstract: Peripheral membrane proteins are ubiquitous throughout cell biology and are required for a variety of cellular processes such as signal transduction, membrane trafficking, and autophagy. Transient ...Peripheral membrane proteins are ubiquitous throughout cell biology and are required for a variety of cellular processes such as signal transduction, membrane trafficking, and autophagy. Transient binding to the membrane has a profound impact on protein function, serving to induce conformational changes and alter biochemical and biophysical parameters by increasing the local concentration of factors and restricting diffusion to two dimensions. Despite the centrality of the membrane in serving as a template for cell biology, there are few reported high-resolution structures of peripheral membrane proteins bound to the membrane. We analyzed the utility of lipid nanodiscs to serve as a template for cryo-EM analysis of peripheral membrane proteins. We tested a variety of nanodiscs and we report a 3.3 Å structure of the AP2 clathrin adaptor complex bound to a 17-nm nanodisc, with sufficient resolution to visualize a bound lipid head group. Our data demonstrate that lipid nanodiscs are amenable to high-resolution structure determination of peripheral membrane proteins and provide a framework for extending this analysis to other systems.
History
DepositionMar 7, 2023-
Header (metadata) releaseJul 12, 2023-
Map releaseJul 12, 2023-
UpdateJul 19, 2023-
Current statusJul 19, 2023Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_29977.png

Downloads & links

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Map

FileDownload / File: emd_29977.map.gz / Format: CCP4 / Size: 103 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationFull map
Voxel sizeX=Y=Z: 0.88 Å
Density
Contour LevelBy AUTHOR: 0.1
Minimum - Maximum-0.1008565 - 0.32685855
Average (Standard dev.)0.00040809295 (±0.011111279)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions300300300
Spacing300300300
CellA=B=C: 264.0 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_29977_msk_1.map
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Additional map: deepEMhancer sharpened map

Fileemd_29977_additional_1.map
AnnotationdeepEMhancer sharpened map
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Additional map: Map for coloring by local resolution

Fileemd_29977_additional_2.map
AnnotationMap for coloring by local resolution
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Additional map: Map filtered by local resolution

Fileemd_29977_additional_3.map
AnnotationMap filtered by local resolution
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Additional map: cryoSPARC sharpened map

Fileemd_29977_additional_4.map
AnnotationcryoSPARC sharpened map
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Half map: Half map A

Fileemd_29977_half_map_1.map
AnnotationHalf map A
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Half map: Half map B

Fileemd_29977_half_map_2.map
AnnotationHalf map B
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Sample components

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Entire : AP2 bound to MSP2N2 nanodisc

EntireName: AP2 bound to MSP2N2 nanodisc
Components
  • Complex: AP2 bound to MSP2N2 nanodisc
    • Protein or peptide: AP-2 complex subunit alpha-2
    • Protein or peptide: AP-2 complex subunit beta
    • Protein or peptide: AP-2 complex subunit mu
    • Protein or peptide: AP-2 complex subunit sigma
    • Protein or peptide: MSP1

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Supramolecule #1: AP2 bound to MSP2N2 nanodisc

SupramoleculeName: AP2 bound to MSP2N2 nanodisc / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Details: Nanodiscs were assembled with a lipid mixture containing 75 mol% DOPC, 15 mol% DOPS, 10 mol% PIP2. Complex was formed by co-elution via gel filtration chromatography.
Source (natural)Organism: Mus musculus (house mouse)
Molecular weightTheoretical: 203.98 KDa

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Macromolecule #1: AP-2 complex subunit alpha-2

MacromoleculeName: AP-2 complex subunit alpha-2 / type: protein_or_peptide / ID: 1 / Enantiomer: LEVO
Source (natural)Organism: Mus musculus (house mouse)
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MPAVSKGDGM RGLAVFISDI RNCKSKEAEI KRINKELANI RSKFKGDKAL DGYSKKKYVC KLLFIFLLGH DIDFGHMEAV NLLSSNRYTE KQIGYLFISV LVNSNSELIR LINNAIKNDL ASRNPTFMGL ALHCIANVGS REMAEAFAGE IPKILVAGDT MDSVKQSAAL ...String:
MPAVSKGDGM RGLAVFISDI RNCKSKEAEI KRINKELANI RSKFKGDKAL DGYSKKKYVC KLLFIFLLGH DIDFGHMEAV NLLSSNRYTE KQIGYLFISV LVNSNSELIR LINNAIKNDL ASRNPTFMGL ALHCIANVGS REMAEAFAGE IPKILVAGDT MDSVKQSAAL CLLRLYRTSP DLVPMGDWTS RVVHLLNDQH LGVVTAATSL ITTLAQKNPE EFKTSVSLAV SRLSRIVTSA STDLQDYTYY FVPAPWLSVK LLRLLQCYPP PEDPAVRGRL TECLETILNK AQEPPKSKKV QHSNAKNAVL FEAISLIIHH DSEPNLLVRA CNQLGQFLQH RETNLRYLAL ESMCTLASSE FSHEAVKTHI ETVINALKTE RDVSVRQRAV DLLYAMCDRS NAQQIVAEML SYLETADYSI REEIVLKVAI LAEKYAVDYT WYVDTILNLI RIAGDYVSEE VWYRVIQIVI NRDDVQGYAA KTVFEALQAP ACHENLVKVG GYILGEFGNL IAGDPRSSPL IQFNLLHSKF HLCSVPTRAL LLSTYIKFVN LFPEVKATIQ DVLRSDSQLK NADVELQQRA VEYLRLSTVA STDILATVLE EMPPFPERES SILAKLKKKK GGSGLEVLFQ

UniProtKB: AP-2 complex subunit alpha-2

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Macromolecule #2: AP-2 complex subunit beta

MacromoleculeName: AP-2 complex subunit beta / type: protein_or_peptide / ID: 2 / Enantiomer: LEVO
Source (natural)Organism: Mus musculus (house mouse)
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MTDSKYFTTN KKGEIFELKA ELNNEKKEKR KEAVKKVIAA MTVGKDVSSL FPDVVNCMQT DNLELKKLVY LYLMNYAKSQ PDMAIMAVNS FVKDCEDPNP LIRALAVRTM GCIRVDKITE YLCEPLRKCL KDEDPYVRKT AAVCVAKLHD INAQMVEDQG FLDSLRDLIA ...String:
MTDSKYFTTN KKGEIFELKA ELNNEKKEKR KEAVKKVIAA MTVGKDVSSL FPDVVNCMQT DNLELKKLVY LYLMNYAKSQ PDMAIMAVNS FVKDCEDPNP LIRALAVRTM GCIRVDKITE YLCEPLRKCL KDEDPYVRKT AAVCVAKLHD INAQMVEDQG FLDSLRDLIA DSNPMVVANA VAALSEISES HPNSNLLDLN PQNINKLLTA LNECTEWGQI FILDCLSNYN PKDDREAQSI CERVTPRLSH ANSAVVLSAV KVLMKFLELL PKDSDYYNML LKKLAPPLVT LLSGEPEVQY VALRNINLIV QKRPEILKQE IKVFFVKYND PIYVKLEKLD IMIRLASQAN IAQVLAELKE YATEVDVDFV RKAVRAIGRC AIKVEQSAER CVSTLLDLIQ TKVNYVVQEA IVVIRDIFRK YPNKYESIIA TLCENLDSLD EPDARAAMIW IVGEYAERID NADELLESFL EGFHDESTQV QLTLLTAIVK LFLKKPSETQ ELVQQVLSLA TQDSDNPDLR DRGYIYWRLL STDPVTAKEV VLSEKPLISE ETDLIEPTLL DELICHIGSL ASVYHKPPNA FVEGSHGIHR K

UniProtKB: AP-2 complex subunit beta

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Macromolecule #3: AP-2 complex subunit mu

MacromoleculeName: AP-2 complex subunit mu / type: protein_or_peptide / ID: 3 / Enantiomer: LEVO
Source (natural)Organism: Mus musculus (house mouse)
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MIGGLFIYNH KGEVLISRVY RDDIGRNAVD AFRVNVIHAR QQVRSPVTNI ARTSFFHVKR SNIWLAAVTK QNVNAAMVFE FLYKMCDVM AAYFGKISEE NIKNNFVLIY ELLDEILDFG YPQNSETGAL KTFITQQGIK SQHQTKEEQS QITSQVTGQI GWRREGIKYR ...String:
MIGGLFIYNH KGEVLISRVY RDDIGRNAVD AFRVNVIHAR QQVRSPVTNI ARTSFFHVKR SNIWLAAVTK QNVNAAMVFE FLYKMCDVM AAYFGKISEE NIKNNFVLIY ELLDEILDFG YPQNSETGAL KTFITQQGIK SQHQTKEEQS QITSQVTGQI GWRREGIKYR RNELFLDVLE SVNLLMSPQG QVLSAHVSGR VVMKSYLSGM PECKFGMNDK IVIEKQGKGT ADETSKS GK QSIAIDDCTF HQCVRLSKFD SERSISFIPP DGEFELMRYR TTKDIILPFR VIPLVREVGR TKLEVKVVIK SNFKPSLL A QKIEVRIPTP LNTSGVQVIC MKGKAKYKAS ENAIVWKIKR MAGMKESQIS AEIELLPTND KKKWARPPIS MNFEVPFAP SGLKVRYLKV FEPKLNYSDH DVIKWVRYIG RSGIYETRC

UniProtKB: AP-2 complex subunit mu

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Macromolecule #4: AP-2 complex subunit sigma

MacromoleculeName: AP-2 complex subunit sigma / type: protein_or_peptide / ID: 4 / Enantiomer: LEVO
Source (natural)Organism: Mus musculus (house mouse)
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString:
MIRFILIQNR AGKTRLAKWY MQFDDDEKQK LIEEVHAVVT VRDAKHTNFV EFRNFKIIYR RYAGLYFCIC VDVNDNNLAY LEAIHNFVE VLNEYFHNVC ELDLVFNFYK VYTVVDEMFL AGEIRETSQT KVLKQLLMLQ SLE

UniProtKB: AP-2 complex subunit sigma

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Macromolecule #5: MSP1

MacromoleculeName: MSP1 / type: protein_or_peptide / ID: 5 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MGHHHHHHIE GRLKLLDNWD SVTSTFSKLR EQLGPVTQEF WDNLEKETEG LRQEMSKDLE EVKAKVQPYL DDFQKKWQEE MELYRQKVEP LRAELQEGAR QKLHELQEKL SPLGEEMRDR ARAHVDALRT HLAPYSDELR QRLAARLEAL KENGGARLAE YHAKATEHLS ...String:
MGHHHHHHIE GRLKLLDNWD SVTSTFSKLR EQLGPVTQEF WDNLEKETEG LRQEMSKDLE EVKAKVQPYL DDFQKKWQEE MELYRQKVEP LRAELQEGAR QKLHELQEKL SPLGEEMRDR ARAHVDALRT HLAPYSDELR QRLAARLEAL KENGGARLAE YHAKATEHLS TLSEKAKPAL EDLRQGLLPV LESFKVSFLS ALEEYTKKLN TQ

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration1 mg/mL
BufferpH: 7.4 / Details: 20 mM HEPES pH 7.4, 100 mM NaCl
GridModel: Quantifoil R1.2/1.3 / Material: COPPER / Mesh: 300 / Pretreatment - Type: PLASMA CLEANING / Pretreatment - Atmosphere: OTHER / Details: TergeoEM Plasma Cleaner
VitrificationCryogen name: ETHANE-PROPANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV
DetailsNanodiscs were assembled with a lipid mixture containing 75 mol% DOPC, 15 mol% DOPS, 10 mol% PIP2. Complex was formed by co-elution via gel filtration chromatography.

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Electron microscopy

MicroscopeFEI TALOS ARCTICA
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 100.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Nominal defocus max: 2.5 µm / Nominal defocus min: 0.5 µm / Nominal magnification: 45000
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Average electron dose: 55.0 e/Å2
Experimental equipment
Model: Talos Arctica / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 1100000
Details: General model gmodel_phosnet_202005_N63_c17.h5 used for crYOLO picking.
Startup modelType of model: OTHER / Details: Ab initio model generation in cryoSPARC v3.2
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
Final reconstructionAlgorithm: BACK PROJECTION / Resolution.type: BY AUTHOR / Resolution: 4.2 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 3.2) / Details: Non-uniform refinement in cryoSPARC v3.2 / Number images used: 148341
FSC plot (resolution estimation)

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